Tetraploid strains of Saccharomyces cerevisiae carrying different dosages of the CYR1+ gene have been constructed. Adenylate cyclase activity observed in these tetraploid strains was proportional to the dosage of the active CYR1+ gene. Of the 57 mutants requiring adenosine 3',5'-monophosphate for growth at 35 degrees C, two allelic temperature-sensitive cyr1 mutants produced thermolabile adenylate cyclase. Crude extract and plasma membrane fraction of cyr1 mutant cells had no adenylate cyclase activity when assayed with GTP or 5'-guanylyl imidodiphosphate in the presence of Mn2+ or Mg2+. Plasma membrane and Lubrol-soluble plasma membrane fractions obtained from the temperature-sensitive cyr1 mutant were thermolabile compared with those from the wild-type strain. Three cyr1 mutants carried nonsense mutations susceptible to ochre (UAA) suppressors, SUP3 and SUP-o, and had no detectable level of adenylate cyclase activity. It is concluded that the cyr1 mutants carry lesions in the structural gene for adenylate cyclase.